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Mixed hind-limb muscle was fractionated as previously described (12), with the following modifications. Homogenates were centrifuged at 1,000 g (pellet), then at 16,000 g to remove mitochondria and plasma membranes yielding the cytosolic fraction as the supernatant. The pellet was then resuspended in 2 ml of homogenization buffer, filtered using a 40-μm nylon mesh cell filter (Fisher) to remove unbroken cells/debris. The filtrate was again centrifuged at 1,000 g, yielding a nuclear fraction that was resuspended in RIPA buffer (Millipore) for Western analysis.
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